The dopamine transporter (DAT) is the primary system responsible for clearance of extracellular dopamine from the synaptic space. As such, it performs a key role in terminating synaptic transmission and in regulating the concentration of dopamine available for binding to pre- and post-synaptic dopamine receptors. DATs undergo protein kinase C mediated phosphorylation and display concomitant reductions in dopamine transport, suggesting that DATs undergo phosphorylation-induced functional regulation. This would provide the neuron with a mechanism for fine temporal and spatial control of extracellular dopamine concentrations, and subsequent downstream dopaminergic neural activity. DAT phosphorylation therefore has the potential to profoundly influence normal dopaminergic neurophysiology, and may be related to mechanisms of dopaminergic neurode.qeneration or abuse of psychostimulants such as cocaine, amphetamine, and methamphetamine. Current evidence suggests that PKC-mediated regulation of DAT occurs by modulation of intracellular traffickinq and control of DAT copy numbers at the plasma membrane, but a definitive relationship between DAT phosphorylation and trafficking has not been established. This study proposes te thoroughly characterize DAT phosphorylation properties and define the relationship between DAT phosphorylation and functional regulation. The long term goal of this research is to understand the physiological significance of DAT phosphorylation under conditions of normal neurophysiology and with respect to involvement with drug abuse. The specific aims designed to achieve these goals are: 1. Identify sites of PKC-stimulated phosphorylation on native and expressed DATs using mass spectrometry. 2. Construct mutants with phosphorylation sites changed to non-phosphate acceptors, and examine their intracellu[ar trafficking. 3. Characterize the ability of substrates and psychostimulants such as methamphetamine and cocaine to affect DAT phosphorylation. 4. Identify the endogenous pathways responsible for physiological control of DAT phosphorylation.